Protein degradation is important for cell cycle control, signal transduction and cell growth. Abnormal protein degradation has been implicated in metabolic disorders, cancer development and muscular dystrophy. A novel pathway of protein degradation in the yeast vacuole has been established in our lab. They key gluconeogenic enzyme, fructose-1.6- biophosphatase (FBPase), is targeted from the cytosol to the yeast lysosome (vacuole) for degradation when Saccharomyces cerevisiae are replenished with glucose. Our long term goal is to understand the FBPase degradation pathway. We have reconstituted this glucose-regulated targeting pathway using semi-intact cells, purified FBPase, an ATP regenerating system and cytosol. FBPase is targeted to the vacuole in the reconstituted system. We have isolated 33 vid (vacuolar import and degradation) mutants defective in the glucose-induced degradation of FBPase. Mutant analysis led to the hypothesis that FBPase is targeted from the cytosol to the intermediate vesicle and then the vacuole for degradation. We have purified a novel FBPase-associated vesicle to near homogeneity. We cloned the VID24 gene involved in vesicle targeting to the vacuole. Vid24p is synthesized and localized to the vesicles. Our specific aims are: (1) Reconstitution of FBPase import into the vesicle using the vid24-1 mutant. We will examine whether the imported FTPase is indeed targeted to the intermediate vesicles. We will divide vid1-vid13 which accumulates FBPase in the cytosol into functional subgroups. (2) Cloning of the VID genes. We plan to clone the VID genes using the colony blotting procedure and study the expression and localization of the Vid proteins. As an alternative approach, we will clone the VID15 gene which is tightly linked to the URA3 gene by chromosomal walking. (3) Purification of cytosolic proteins required for FBPase import into the vesicles. We will use the vid1-vid13 mutants that contain defective cytosolic factor(s) and add fractionated wild type cytosol to identify the fractions that complement the mutant cytosol defect. If we identify such protein, we will make mutants and prepare cytosol from the mutants to test whether the cytosol is defective in FBPase import in vitro. We will examine whether FBPase import into the vesicles is regulated by ATP or GTP hydrolysis.